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Nitric Oxide–Dependent Human Acrosomal Loss Induced by PPCM (SAMMA) and by Nitric Oxide Donors Occurs by Independent Pathways: Basis for Synthesis of an Improved Contraceptive Microbicide

ROBERT A. ANDERSON^*,

KENNETH A. FEATHERGILL^*,

CALVIN J. CHANY, II^*,,

SANJAY JAIN^,||

ALEKSEJ KRUNIC

From the ^* Program for the Topical Prevention of Conception and Disease (TOPCAD) and the Department of Obstetrics and Gynecology, Rush University Medical Center, Chicago, Illinois; and the Department of Medicinal Chemistry and Pharmacognosy, University of Illinois at Chicago, Health Sciences Center, Chicago, Illinois.
Present address: Thermo Fisher Scientific, 2555 Kerper Blvd, Dubuque, IA 52002.
^|| Present address: Chembiotek, International Biotech Park, Bio Research Centre, Mulshi, Hinjewadi, Pune 411057, India.

Correspondence to: Dr Robert A. Anderson, Jr, Ob/Gyn Research, Rush Medical Center, Chicago, IL 60612 (e-mail: robertan{at}corecomm.net).

PPCM (previously designated sulfuric acid–modified mandelic acid [SAMMA]) is a contraceptive microbicide in preclinical development. Its contraceptive activity is attributable in part to its ability to promote premature acrosomal loss. Prior studies showed that PPCM-induced human acrosomal loss (PAL) is Ca²⁺-dependent. This study was carried out to determine transduction elements downstream from Ca²⁺ entry. PAL is inhibited by inhibitors selective for endothelial-type nitric oxide synthase. PAL is completely inhibited by 0.1 µM ODQ (soluble guanylate cyclase inhibitor). PAL is inhibited by protein kinase G inhibitors with selectivity for the type II isotype. Several inhibitors of the nitric oxide/cyclic guanosine monophosphate (cGMP)/protein kinase G pathway induce Ca²⁺-dependent acrosomal loss when added alone. These responses are inhibited by nifedipine, a blocker of Ca_v1.x voltage-dependent channels. Acrosomal loss induced by the nitric oxide donor SNAP (SNAL) does not require added Ca²⁺. Sperm production of nitric oxide is increased by PPCM, an effect inhibited by nitro-L-arginine (nitric oxide synthase inhibitor). Although inhibited by ODQ, SNAL and acrosomal loss induced by other nitric oxide donors are unaffected by KT5823 (protein kinase G inhibitor). Unlike PAL, SNAL is partially inhibited by KT5720 (protein kinase A inhibitor) and genistein (protein tyrosine kinase inhibitor). Acrosomal loss response to PPCM and SNAP added in combination suggests that these agents act by independent mechanisms. A PPCM derivative was synthesized, in which a nitric oxide donor was esterified to PPCM (NOSPPA-23). NOSPPA-23 induces acrosomal loss with or without added Ca²⁺. The ED₅₀ of NOSPPA-23 (4.8 nM) in the presence of Ca²⁺ is 35-fold less than that of PPCM. These findings suggest the following: 1) elements responsible for PAL include endothelial nitric oxide synthase, soluble guanylate cyclase, and type II protein kinase G; 2) the resting state of the nitric oxide/cGMP/protein kinase G pathway is a determinant of acrosomal status; 3) PPCM and nitric oxide donors induce acrosomal loss via nitric oxide, but through independent pathways; and 4) covalent attachment of a nitric oxide donor to PPCM provides synergistic efficacy as a stimulus of acrosomal loss. Further studies with this novel prototype as an improved contraceptive microbicide are warranted.

     Key words: Signal transduction, mechanism, nitric oxide synthase, cGMP, guanylate cyclase, protein kinase G, protein kinase A, protein tyrosine kinase